Abstract
Abstract
Mitogen-Activated Protein Kinases (MAPKs) are signalling proteins that are highly conserved among eukaryotes. They are involved in many biological pathways including cell differentiation and response to different stress situations. The MAPK cascade is composed of three signalling kinases, MAP3K, MAP2K and MAPK, which phosphorylate each other in a sequential manner. The different MAPK substrates are regulated by phosphorylation on serine or threonine. This thesis presents a new method for purification of phosphoproteins from a complex protein extract. Subsequent studies using this method identified AtPHOS34 to be phosphorylated upon biotic stress. The phosphorylation site of AtPHOS34 was also determined.
Several Arabidopsis thaliana MPK3/6 in vitro substrates were identified by a peptide and bioinformatics screen and the predicted phosphorylation sites were confirmed with kinase assays using mutated and wild-type proteins. MAPK Substrate 80 (here denoted MKS80) was characterised as an in planta MAPK substrate and in detail analyses of MKS80 linked its function to stomatal patterning and regulation of carbohydrate balance. The MKS80 interaction partner α-glucan phosphorylase (PHS2) was established by a yeast two-hybrid screen, in vitro pull-down and in planta co-immunoprecipitation assays. PHS2 catalyses the reversible reaction between glucose-1-phosphate and highly branched soluble heteroglycans and was shown to be important in senescence. Biochemical analyses revealed a stable level of PHS2 and light-dependent accumulation of MKS80. Arabidopsis over-expressing wild-type MKS80 had a lower starch content, whereas plants over-expressing the phosphomimetic form of MKS80 died at the seedling stage, clearly demonstrating the importance of this phosphorylation. In plants over-expressing MKS80, the anthocyanin level was lowered in different stress situations. Slower senescence was detected in detached leaves over-expressing MKS80, indicating a change in metabolism. A system for delayed senescence can be of interest for the agricultural industry.
Mitogen-Activated Protein Kinases (MAPKs) are signalling proteins that are highly conserved among eukaryotes. They are involved in many biological pathways including cell differentiation and response to different stress situations. The MAPK cascade is composed of three signalling kinases, MAP3K, MAP2K and MAPK, which phosphorylate each other in a sequential manner. The different MAPK substrates are regulated by phosphorylation on serine or threonine. This thesis presents a new method for purification of phosphoproteins from a complex protein extract. Subsequent studies using this method identified AtPHOS34 to be phosphorylated upon biotic stress. The phosphorylation site of AtPHOS34 was also determined.
Several Arabidopsis thaliana MPK3/6 in vitro substrates were identified by a peptide and bioinformatics screen and the predicted phosphorylation sites were confirmed with kinase assays using mutated and wild-type proteins. MAPK Substrate 80 (here denoted MKS80) was characterised as an in planta MAPK substrate and in detail analyses of MKS80 linked its function to stomatal patterning and regulation of carbohydrate balance. The MKS80 interaction partner α-glucan phosphorylase (PHS2) was established by a yeast two-hybrid screen, in vitro pull-down and in planta co-immunoprecipitation assays. PHS2 catalyses the reversible reaction between glucose-1-phosphate and highly branched soluble heteroglycans and was shown to be important in senescence. Biochemical analyses revealed a stable level of PHS2 and light-dependent accumulation of MKS80. Arabidopsis over-expressing wild-type MKS80 had a lower starch content, whereas plants over-expressing the phosphomimetic form of MKS80 died at the seedling stage, clearly demonstrating the importance of this phosphorylation. In plants over-expressing MKS80, the anthocyanin level was lowered in different stress situations. Slower senescence was detected in detached leaves over-expressing MKS80, indicating a change in metabolism. A system for delayed senescence can be of interest for the agricultural industry.
Original language | English |
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Qualification | Doctor |
Awarding Institution |
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Supervisors/Advisors |
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Award date | 2010 Nov 25 |
ISBN (Print) | 978-91-7473-004-3 |
Publication status | Published - 2010 |
Bibliographical note
Defence detailsDate: 2010-11-25
Time: 10:00
Place: the Biology Lecture Hall, Sölvegatan 35, 223 62Lund
External reviewer(s)
Name: Ekengren, Sophia
Title: Dr. Assistant Professor
Affiliation: Department of Botany, Stockholm University, 106 91 Stockholm , Sweden, 08-163754, [email protected]
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The information about affiliations in this record was updated in December 2015.
The record was previously connected to the following departments: Department of Cell and Organism Biology (Closed 2011.) (011002100)
Subject classification (UKÄ)
- Biological Sciences
Free keywords
- PHS2
- stomata
- signalling
- Arabidopsis thaliana
- MKS80
- MAPK